Saturday, April 7, 2018

Optimized collision energy by ID rates!

HCD is awesome. It's super fast. It breaks more-or-less right along the b/y ion backbone that you get from CID and it's super fast. Also -- it's fast.

Unfortunately -- nothing beats CID in terms of predictable fragmentation. HCD fragmentation efficiency can vary quite a bit (look a the actual eV used in your scan header when you're using normalized CE for proof of this) and is dependent on some additional variables like the fragmentation energy calibration, your HCD gas pressure (and -- no proof of this yet -- the quality of the N2 being delivered.)

I think I've rambled on here more than once about my love of the PROCAL peptide standard. One reason for that is it gives you the capability of calibrating your HCD CE so that your fragmentation patterns match between instruments (PROCAL paper here).

It's more complicated than you'd guess, unfortunately. But this team sets up a really nice mechanism for determining it. They use a series of different fragmentation energies versus Mascot scores and other metrics and work out this multiphase ideal shown above. There is some further interesting info in here, including how to strengthen different ion series --- super important if you're thinking about doing something crazy like studying peptides where the y-ions aren't going to be as helpful to you.